Method of producing infectious reovirus

ABSTRACT

A simple and efficient method of producing mammalian reovirus is developed using HEK 293 cells. The method provides for fast production of reovirus in high yield. Furthermore, this method provides for a simpler purification procedure of the produced reovirus.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 60/224,026, filed Aug. 10, 2000, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to a method of producing infectious mammalianreovirus which is suitable for clinical administration to mammals,including human beings.

REFERENCES

U.S. Pat. No. 5,023,252.

Berry et al., Biotechnology and Bioengineering, “Production of ReovirusType-1 and Type-3 from Vero Cells Grown on Solid and MacroporousMicrocarriers”, Biotechnology and Bioengineering 62: 12-19 (1999).

Bos, J. L., “Ras Oncogenes in Human Cancer: A Review”, Canc. Res.49(17): 4682-4689 (1989).

Chandron and Nibert, “Protease cleavage of reovirus capsid protein mu1and mu1C is blocked by alkyl sulfate detergents, yielding a new type ofinfectious subvirion particle”, J. of Virology 72(1):467-75 (1998).

Coffey, M. C., et al., “Reovirus therapy of tumors with activated Raspathway”, Science 282: 1332-1334 (1998).

Davis, et al., Microbiology, Lippincott, Philadelphia (1990).

Fields, B. N. et al., Fundamental Virology, 3rd Edition,Lippincott-Raven (1996).

Japanese Patent 63044532A, published Feb. 25, 1988.

McRae, M. A. and Joklik, W. K., “The nature of the polypeptide encodedby each of the 10 double-stranded RNA segments of reovirus type 3”,Virology, 89:578-593 (1979).

Nibert et al., “Reovirus and their replication”, in Fields et al.,Fundamental Virology, 3rd Edition, Lippincott-Raven (1996).

Smith, R. E., et al., “Polypeptide components of virions, top componentand cores of reovirus type 3”, Virology, 39:791-800 (1969).

Strong, J. E. and P. W. Lee, “The v-erbV oncogene confers enhancedcellular susceptibility to reovirus infection”, J. Virol. 70: 612-616(1996).

Strong, J. E., et al., “Evidence that the Epidermal Growth FactorReceptor on Host Cells Confers Reovirus Infection Efficiency”, Virology197(1): 405-411 (1993).

Strong, J. E., et al., “The molecular basis of viral oncolysis:usurpation of the Ras signaling pathway by reovirus”, EMBO J. 17:3351-3362 (1998).

Taber et al., “The selection of virus-resistant Chinese hamster ovarycells”, Cell 8: 529-533 (1976).

WO99/08692A1, published Feb. 25, 1999.

All of the above publications, patents and patent applications areherein incorporated by reference in their entirety to the same extent asif the disclosure of each individual publication, patent application orpatent was specifically and individually indicated to be incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Reovirus is a double-stranded RNA virus with a segmented genome. Thereceptor for the mammalian reovirus, sialic acid, is a ubiquitousmolecule, therefore reovirus is capable of binding to a multitude ofcells. However, most cells are not susceptible to reovirus infection andbinding of reovirus to its cellular receptor results in no viralreplication or virus particle production. This is probably the reasonwhy reovirus is not known to be associated with any particular disease.

It was discovered recently that cells transformed with the ras oncogenebecome susceptible to reovirus infection, while their untransformedcounterparts are not (Strong et al., 1998). For example, whenreovirus-resistant NIH 3T3 cells were transformed with activated Ras orSos, a protein which activates Ras, reovirus infection was enhanced.Similarly, mouse fibroblasts that are resistant to reovirus infectionbecame susceptible after transfection with the EGF receptor gene or thev-erbB oncogene, both of which activate the ras pathway (Strong et al.,1993; Strong et al., 1996). Thus, reovirus can selectively infect andreplicate in cells with an activated Ras pathway.

The ras oncogene accounts for a large percentage of mammalian tumors.Activating mutations of the ras gene itself occur in about 30% of allhuman tumors (Bos, 1989), primarily in pancreatic (90%), sporadiccolorectal (50%) and lung (40%) carcinomas, as well as myeloid leukemia(30%). Activation of factors upstream or downstream of ras in the raspathway is also associated with tumor. For example, overexpression ofHER2/Neu/ErbB2 or the epidermal growth factor (EGF) receptor is commonin breast cancer (25-30%), and overexpression of platelet-derived growthfactor (PDGF) receptor or EGF receptor is prevalent in gliomas andglioblastomas (40-50%). EGF receptor and PDGF receptor are both known toactivate ras upon binding to their respective ligand, and v-erbB encodesa constitutively activated receptor lacking the extracellular domain.

Since a large number of human tumors are accounted for by geneticalteration of the proto-oncogene ras or a high Ras activity, reovirustherapy is a new, promising therapy for such conditions (Coffey et al.,1998). Reovirus therapy is highly selective for Ras-associated tumorcells and leaves normal cells uninfected. Consequently, a simple andcost-effective method for the production of infectious reovirus suitablefor clinical administration in human beings is needed.

Because reovirus does not pose a serious threat to human health, therehas not been an intensive effort to produce reovirus efficiently. Themammalian reovirus is traditionally grown in mouse L-929 fibroblasts(Nibert et al., 1996). It has also been reported to grow in Chinesehamster ovary cells and Vero cells, an African green monkey kidney cellline (Taber et al., 1976; Davis et al., 1990). In addition, a primaryculture of swine kidney was used to culture a swine reovirus (JapanesePatent 63044532A, published Feb. 25, 1988). In a study aiming at massproduction of the reovirus, Berry et al. conducted an investigation ofthe optimal methods of culturing Vero cells and the subsequent reovirusinfection (Berry et al., 1999). Vero cells were grown in eitherCytodex-1 or Cultispher-G microcarriers, and culture parameters such ascell density, time course of viral growth and the ratio of cells tobeads in the microcarrier were varied and virus yield determined. Thestudy showed that the yield of virus varied greatly with the cultureparameters, and complicated culture conditions (e.g. cell number perbeads relative to multiplicity of infection) were required to obtainreasonable yield. Therefore, there remains a need for a simple,efficient method to produce clinically useful reovirus.

SUMMARY OF THE INVENTION

The present invention is directed to a simple and efficient method ofproducing reovirus by culturing reovirus in human embryo kidney 293 (HEK293) cells. The yield in HEK 293 cells is unexpectedly high,particularly in comparison to previous work using L-929 cells or Verocells. Moreover, by using the present invention, a high yield isachieved early in the course of infection, before viral particles arereleased from the cells. Therefore, the virus can be harvested earlyfrom intact cells, allowing the virus to be purified in the absence ofthe culture media. This makes the purification procedure relativelysimple.

Accordingly, one aspect of the invention provides a method of producingmammalian reovirus, comprising the steps of contacting HEK 293 cellswith a mammalian reovirus under conditions which result in reoviralinfection of said HEK 293 cells; incubating the culture of said infectedcells for a period of time sufficient to allow for viral replication;and harvesting the virus produced.

Any mammalian reovirus can be produced using this method. In particular,human reovirus for clinical administration in human beings can beproduced using this method. Most particularly, reovirus serotype 3 andthe Dearing strain are produced. Other mammalian reovirus can beproduced efficiently by the presently claimed method as well.

Another feature of the invention is that because reovirus is producedrapidly in the HEK 293 cells, this invention provides for a fast andcost-effective method of producing reovirus. Also, since virus titer inthe HEK 293 cell culture is high before the cells are completely lysed,the virus may be harvested when it is still associated with the cell,hence the purification procedure of the virus can be relatively simple,further reducing the cost of production. Therefore, this inventionprovides for a method wherein the virus is harvested before all thecells are lysed. Preferably, the virus is harvested when 20-95% of thecells remain viable, more preferably 35-90%, and most preferably 50-80%.

Another aspect of the invention is that a wide range of multiplicity ofinfection may be used to achieve virus production. Although amultiplicity of infection of 0.1 is sufficient, higher multiplicitiescan be used to shorten the production time, e.g., a multiplicity of upto 10. Preferably a multiplicity of infection of less than 1 is used,more preferably at or around 0.5.

Another aspect of the invention provides for a method of producingreovirus in both adherent and suspension cell cultures, since HEK 293cells can be adapted to grow in both kinds of cultures. HEK 293 cellswhich have been adapted for other purposes or culture conditions, ortransformed with an exogenous DNA, are also useful in the presentinvention.

The present invention also provides for a mammalian reovirus compositionwhich is prepared by the methods described above. This composition maypreferably be purified. The composition is suitable for extendedstorage, e.g. by freezing. The composition is useful to treat tumors orother cell proliferative disorders caused by an activated ras pathway,and is suitable for administration in human beings because HEK 293 cellsare approved for growing biological products by the Food and DrugAdministration. The composition can further comprise a pharmaceuticallyacceptable carrier or excipient, and/or a therapeutical agent which canbe used along with the reovirus for its intended purposes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of producing mammalianreovirus by using human embryo kidney 293 (HEK 293) cells. We found thatHEK 293 cell are a more efficient host cell for reovirus than L-929cells or Vero cells. Although never reported to support reovirusreplication before, HEK 293 cells produced more reovirus in a shortertime course than L-929 cells or Vero cells. Consequently, by using HEK293, production costs can be lowered. Moreover, because the reovirus isstill associated with HEK 293 cells when the titer is sufficiently high,a relatively simple procedure may be used to purify the virus andfurther lower the production cost.

Prior to describing the invention in further detail, the terms used inthis application are defined as follows unless otherwise indicated.

Definitions

As used herein, “HEK 293 cells” refer to the human embryo kidney cellline designated 293 (ATCC Number CRL-1573) or its derivatives. Forexample, 293/SF cells (ATCC Number CRL-1573.1) are HEK 293 cells whichhave been adapted to grow in serum-free media. Also contemplated in thisinvention are HEK 293 cells adapted to grow in other culture conditions,or any kind of HEK 293 cells or derivatives which are transformed withan exogenous DNA, provided that this transformation does not impair theability of the cells to support efficient reovirus production asdescribed in this invention.

As used herein, “reovirus” refers to any virus classified in thereovirus genus, whether naturally occurring, modified or recombinant.Reoviruses are viruses with a double-stranded, segmented RNA genome. Thevirions measure 60-80 nm in diameter and possess two concentric capsidshells, each of which is icosahedral. The genome consists ofdouble-stranded RNA in 10-12 discrete segments with a total genome sizeof 16-27 kbp. The individual RNA segments vary in size. Three distinctbut related types of reovirus have been recovered from many species. Allthree types share a common complement-fixing antigen.

The human reovirus consists of three serotypes: type 1 (strain Lang orT1L), type 2 (strain Jones, T2J) and type 3 (strain Dearing or strainAbney, T3D). The three serotypes are easily identifiable on the basis ofneutralization and hemagglutinin-inhibition assays (see, for example,Fields, B. N. et al., 1996).

The reovirus may be naturally occurring or modified. The reovirus is“naturally-occurring” when it can be isolated from a source in natureand has not been intentionally modified by humans in the laboratory. Forexample, the reovirus can be from a “field source”, that is, from ahuman who has been infected with the reovirus.

The reovirus may be modified but still capable of lytically infecting amammalian cell having an active ras pathway. The reovirus may bechemically or biochemically pretreated (e.g., by treatment with aprotease, such as chymotrypsin or trypsin) prior to administration tothe proliferating cells. Pretreatment with a protease removes the outercoat or capsid of the virus and may increase the infectivity of thevirus. The reovirus may be coated in a liposome or micelle (Chandron andNibert, 1998). For example, the virion may be treated with chymotrypsinin the presence of micelle forming concentrations of alkyl sulfatedetergents to generate a new infectious subvirion particle.

The reovirus may be a recombinant (i.e. reasserted) reovirus from two ormore types of reoviruses with differing pathogenic phenotypes such thatit contains different antigenic determinants, thereby reducing orpreventing an immune response by a mammal previously exposed to areovirus subtype. Such recombinant virions can be generated byco-infection of mammalian cells with different subtypes of reovirus withthe resulting resorting and incorporation of different subtype coatproteins into the resulting virion capsids.

As used herein, “contacting” a cell with a virus refers to placing thevirus in the culture of the cell such that the virus has the opportunityto make a contact with the cell, which may lead to successful infectionby the virus.

As used herein, “viral infection” refers to the entry of a virus into acell and the subsequent replication of the virus in the cell.

As used herein, “multiplicity of infection” refers to the ratio of thenumber of virus to the number of cells when a virus is used to contactcells.

As used herein, “cell lysis” refers to the disruption of cell membraneof a cell and the subsequent release of all or part of the content ofthe cell.

As used herein, “complete lysis” refers to the lysis of every cell in aculture of multiple cells.

As used herein, “culture conditions” refer to the conditions used in acell culture, including but not limited to the temperature, type ofculture containers, humidity, concentration of CO₂ or any other gas usedin the culture containers, type of the culture medium, the initialdensity of the cultured cells, and if the cells are infected with avirus, the initial multiplicity of infection.

As used herein, a virus that is “cell associated” refers to a viruswhich is attached to or trapped in part of a cell in which the virus hasbeen produced. Thus, a virus is cell associated before the host cell islysed. When cell lysis begins, a virus may be still attached to ortrapped in part of the broken cell and remain cell associated. However,when the virus is released free into the medium, it is not cellassociated anymore.

As used herein, a cell is “disrupted” when the cell membrane is rupturedand at least some of the cell content is released from the cell. A cellmay be disrupted, for example, by freeze-thawing, sonication ordetergent treatments.

As used herein, “harvest” the virus refers to the act of collecting theproduced virus from a cell culture which has been previously infectedwith the virus. Harvesting of the virus may involve breaking up the hostcell if the virus is still cell associated. Alternatively but lesspreferably, viral particles which have been released into the culturemedia can be harvested from the media.

As used herein, “cytopathic effect” is indicated by the cells becomingswollen and granular in appearance and the cell clumps breaking up. Thecells which show a cytopathic effect stain negative in a viable cellcount because they will take up the staining dye.

As used herein, “adherent cells” refer to cells which adhere to theculture containers in a cell culture. Examples of adherent cells includemonolayer cells, which are cells that form a single layer of cells onthe surface of a culture container. “Suspension cells” or “suspendedcells” refer to cells which do not adhere to culture containers in acell culture. Suspension cells can be grown in a “spin culture”, whichis a culture in which the culture medium is stirred continuously duringthe culture process.

As used herein, “viability of the cells” or “percentage of cellsremaining viable” is the percentage of the cells which do not show acytopathic effect in a population.

As used herein, “harvest time” refers to the time point at which thereovirus is collected and purified. The virus is preferably harvestedwhen titer is sufficiently high and the virus is still cell-associated.Although the virus may be harvested even after complete cell lysis hasoccurred, it is desirable to harvest the virus before it is releasedfrom the cells to simplify the purification process. Thus, viability ofthe cells is routinely measured as an indication of whether the virus isstill cell-associated. The virus is generally harvested when at least 5%of the cells are viable. Preferably, the virus is harvested when 20-95%of the cells are viable, more preferably when 35-90% cells remainviable, and most preferably when 50-80% cells remain viable.

Methods

Normal cells are generally not susceptible to reovirus infection, andcultured cell lines vary to a great extent in their ability to supportreovirus production. In our attempt to develop an efficient host cell toproduce reovirus, a variety of cells were employed and HEK 293 cellsproved to be very efficient. In a typical experiment (Example 1), HEK293, Vero and L-929 cells were grown to confluency and infected with thereovirus at a multiplicity of infection (m.o.i.) of 1. The yield ofvirus was determined at various time post infection. Surprisingly, HEK293 cells, which have not been reported to support reovirus growth,produced almost 50 times more reovirus at 24 hours post infection thanL-929 cells, which are routinely used to culture mammalian reovirus.Vero cells produced even less reovirus at this point, yielding 3000times less reovirus than the HEK-293 cells.

At 36-48 hours post infection, the virus yield in the HEK-293 cellsbegan to plateau, but the titer was still one order of magnitude higherthan the titer produced in L-929 cells and two orders of magnitudehigher than that of Vero cells. It was not until 96 hours post infectionthat all three cells lines produced about the same titer of reovirus, at10⁹ to 10¹⁰ per milliliter.

These results indicate that the HEK-293 cell is a very efficient systemfor the production of reovirus, allowing for shortened production timewhich will markedly reduce the cost of production.

To further optimize the HEK 293 cell production conditions, reovirus wasused to infect the HEK 293 cells at various m.o.i., and the yield wasdetermined (Example 2). The results suggest that a lower m.o.i. is evenmore advantageous. Thus, at 48 hours post infection, the cells whichwere inoculated at a m.o.i. of 0.5 produced more than 10¹⁰ viruses perml, which was the maximal yield at these culture conditions. After thispoint, the titer went down by about two fold, and reached the maximalyield again at 96 hours. A similar pattern was observed for the culturewith an initial m.o.i. of 0.1. Although the reason is not clear, it ispossible that the decrease at 72 hours is caused by proteolyticdegradation of the virus. This is apparently followed by replication toproduce the higher yield at 96 hours.

Consequently, the best time to harvest reovirus under these cultureconditions is 36-60 hours post infection. At this period of time, thetiter is high, and the virus is still associated with the cell fragmentsand membranes, which makes purification of the virus relatively simple.At 96 hours, all the cells have lysed and the virus is released into themedia along with the degradation products of the dead cells, makingpurification much more complicated than when the virus is cellassociated.

For best efficiency, the virus should be harvested when the yield issufficiently high but most of the virus is still associated with thecells. The harvest time should be determined empirically when cultureconditions are varied. To determine if the virus is associated with thecells, a small aliquot of the culture can be examined, e.g., under themicroscope, to determine the degree of cell viability at different timepoints after infection. Alternatively, a viable stain can be conductedto determine the percentage of viable cells. To simplify thepurification process, the virus is typically harvested before all thecells have been lysed. Preferably, the virus is harvested when 20-95% ofthe cells remain viable. More preferably, the virus is harvested when35-90%, and most preferably 50-80%, of the cells remain viable.

HEK 293 cells are adherent cells and can be grown in cell cultureflasks, roller bottles, microcarrier systems or hollow fiber systems, orany other system that is suitable for growing adherent cells. HEK 293cells may be modified to generate derivative cells. For example, the293/SF cell (ATCC Number CRL-1573.1) was derived from the HEK 293 celland adapted to serum-free culture conditions. The 293/SF cells grow as amixture of adherent and suspension cells and may be grown in any of theculture containers described above, as well as spinner bottles, stirredvessels (fermenters), hollow fiber systems, or any other culturecontainers suitable for suspension cells.

In order to produce industrial amounts of reovirus, 293/SF cells werecultured in 15 L spinner flasks and infected with reovirus at amultiplicity of infection of 0.5 when cell density reached 10⁶/ml. Theculture was incubated until cell lysis began, as evidenced by theculture media color change from red to orange due to the presence ofPhenol Red in the media, or by a viable cell count under the microscope.At this point, the virus was harvested by centrifugation. The virus wasthen purified as described in Materials and Methods and used in clinicaladministrations or stored for future use. For storage, the virus can befrozen or lyophilized according to methods established in the art, withor without stabilizing agents.

Reovirus Compositions

Reovirus compositions may be prepared using reovirus produced by themethods described above. The compositions are suitable for clinicaladministration in mammals, including human beings. When used forclinical administrations, the compositions are preferably purified.Human reovirus, particularly serotype 3, most particularly strainDearing, is the preferred composition prepared by the methods of thisinvention. However, other mammalian reovirus may be produced as well.

This invention also includes pharmaceutical compositions which containone or more of the reoviruses, made according to the presentapplication, associated with “pharmaceutically acceptable carriers orexcipients”. In making the compositions of this invention, the activeingredient/reovirus is usually mixed with an excipient, diluted by anexcipient or enclosed within such a carrier which can be in the form ofa capsule, sachet, paper or other container. When the pharmaceuticallyacceptable excipient serves as a diluent, it can be a solid, semi-solid,or liquid material, which acts as a vehicle, carrier or medium for theactive ingredient. Thus, the compositions can be in the form of tablets,pills, powders, lozenges, sachets, cachets, elixirs, suspensions,emulsions, solutions, syrups, aerosols (as a solid or in a liquidmedium), ointments containing, for example, up to 10% by weight of theactive compound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, and sterile packaged powders.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

For preparing solid compositions such as tablets, the principal activeingredient/reovirus is mixed with a pharmaceutical excipient to form asolid preformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedherein. Preferably the compositions are administered by the oral ornasal respiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner.

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the reovirus of the present invention in controlled amounts.The construction and use of transdermal patches for the delivery ofpharmaceutical agents is well known in the art. See, for example, U.S.Pat. No. 5,023,252, herein incorporated by reference. Such patches maybe constructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents.

Other suitable formulations for use in the present invention can befound in Remington's Pharmaceutical Sciences.

The following examples are offered to illustrate this invention and arenot to be construed in any way as limiting the scope of the presentinvention.

EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. Abbreviations not defined have their generally acceptedmeanings.

CI=Confidence Interval

TCID₅₀=Tissue Culture Infectious Dose₅₀

μM=micromolar

mM=millimolar

M=molar

ml=milliliter

μl=microliter

mg=milligram

μg=microgram

g/L=grams per liter

rpm=revolutions per minute

FBS=fetal bovine serum

DTT=dithiothrietol

NP-40=Nonidet P-40 (Octylphenoxy Polyethoxy Ethanol)

SDS=sodium dodecyl sulfate

PBS=phosphate buffered saline

β-ME=β-mercaptoethanol

MOI or m.o.i.=multiplicity of infection

PFU=plaque forming units

hr=hour

° C.=degree Celsius

General Method

Cells and Virus

Human embryo kidney 293 (HEK 293), Vero (African green monkey kidney)cells, and mouse fibroblast L-929 cells were provided by themanufacturer BioReliance Corporation (Rockville, Maryland). HEK 293cells were grown in a culture medium containing 10% heat-inactivatedhorse serum and 90% of the following mixture: Eagle's minimum essentialmedium with 2 mM L-glutamine and Earle's Balanced Salt Solution adjustedto contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids,and 1.0 mM sodium pyruvate. Mouse L-929 and Vero cells were propagatedin a culture medium containing 10% FBS and 90% of the following mixture:Eagle's minimum essential medium with 2 mM L-glutamine and Earle'sBalanced Salt Solution adjusted to contain 1.5 g/L sodium bicarbonate,0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate.

The 293/SF cells were grown in 293 Serum Free Medium (Life Technologies,Rockville, Maryland) supplemented with 4 mM L-glutamine at 36° C.±2 °C., 6%±2% CO₂ and 80%±5% relative humidity in spinner flasks at animpeller speed of 35-40 rpm.

The Dearing strain of reovirus serotype 3 used in these studies waspropagated in suspension cultures of L-929 cells purified according toSmith (Smith et al., 1969) with the exception that β-mercaptoethanol(β-ME) was omitted from the extraction buffer. Reovirus labelled with[³⁵S]-methionine was grown and purified as described by McRae and Joklik(McRae and Joklik, 1979). The particle/PFU ratio for purified reoviruswas typically 100/1.

Infection of Monolayer Cells and Quantitation of Virus

Confluent monolayers of HEK 293, Vero, and L-929 cells were grown in24-well plates and infected with a reovirus at known multiplicities ofinfection. After 1 hr incubation at 37° C., the monolayers were washedwith warm media and then incubated in their culture medium. At varioustime points postinfection, a mixture of NP-40 and sodium deoxycholatewas added directly to the media on the infected monolayers to finalconcentrations of 1% and 0.5%, respectively. The lysates were thenharvested and virus yields were determined by plaque titration on L-929cells and expressed as Log₁₀TCID₅₀/ml.

Infection of Suspension Cells and Purification of Virus

293/SF cells were grown to 10⁶/ml and infected with the reovirus. Theculture was allowed to grow until the color of the medium turned fromred to orange, or until the viability of the cells dropped to thedesired level as evidenced by a viable cell count. Viable cell countscan be performed under the microscope for cells that do not show acytopathic effect, which is indicated by the cells becoming swollen andgranular in appearance and the cell clumps breaking apart. Viable cellcounts can also be performed by a viable stain as commonly used in theart. When the desired cell viability level was reached, the cells werepelleted in a centrifuge and resuspended in 10 mM Tris, pH 7.4, 250 mMNaCl and 0.1% Triton X-100.

The cells were then lysed by freeze-thawing and kept on ice for 20-40minutes with periodical vortexing to mix and lyse the cells. Thesuspension was extracted with an equal volume of pre-chilled Freon®(1,1,2-trichloro-1,1,2-trifluoro-ethane) by vortexing for 10 minutes,followed by centrifugation at 2500 rpm for 10 minutes at 4° C. toseparate the difference phases. The aqueous (top) phase was removed andre-extracted twice as described above. After the final extraction, theaqueous layer was transferred to a fresh tube, and Triton X-100 wasadded to a final concentration of 0.5%.

The virus was purified by a cesium chloride step gradient. The gradientcontained two layers of CsCl solutions (1.20 g/ml and 1.4 g/ml,respectively) prepared in 10 mM Tris (pH 7.4). The virus suspension wasloaded on top of the gradient and centrifuged in a SW 28.1 rotor at26,000 rpm for 2 hours at 4° C. The viral band (the lower of the twobands because the upper band contained empty capsids) was harvested anddialyzed against sterile PBS.

Example 1 Determination of Optimal Cell Lines for the Production ofReovirus

To determine whether there were differences between susceptible celllines in the amount of reovirus produced as a consequence of infection,a number of different cell lines that exhibited reovirus susceptibilitywere assayed for relative amount of reovirus produced. Of particularinterest were those cell lines that had been approved by variousregulatory authorities for the production of a biological agent.Accordingly, HEK 293, Vero, and L-929 cells were exposed to reovirus,and their ability to produce reovirus was compared.

Quantitation of viral production was accomplished by harvesting infectedcells and their growth media at various time points post infection. Thelysates produced were subsequently subjected to plaque titrationanalysis to determine the viral yield, which is expressed as titer ±95%CI (Log₁₀TCID₅₀/ml) in Table 1 below. The results indicate that, whereasall of the tested cells were susceptible to reovirus infection, therewere considerable differences in the amount of virus produced in each ofthese cells lines.

TABLE 1 Time Course of Viral Yield of Various Cell Lines (expressed astiter ± 95% CI in Log₁₀TCID₅₀/ml) TIME HEK 293 Vero L929 24 HOURS 8.30 ±0.51 4.80 ± 0.35 6.68 ± 0.24 36 HOURS 9.05 ± 0.43 5.55 ± 0.32 7.93 ±0.40 48 HOURS 9.55 ± 0.49 6.68 ± 0.40 8.55 ± 0.49 72 HOURS 9.30 ± 0.438.18 ± 0.36 9.05 ± 0.32 96 HOURS 9.80 ± 0.35 9.93 ± 0.59 9.30 ± 0.43

The results show clearly that the amounts of virus produced were highestin the HEK 293 cells. Further, the HEK 293 cells produced more virusearlier, allowing for shortened production times for the manufacture ofthe reovirus.

Example 2 Effect of Starting Multiplicity of Infection on Final ViralProduction

To determine whether the starting multiplicity of infection determinesfinal viral output, HEK 293 cells were infected with a range of startingmultiplicities of infection (m.o.i.) between 1 and 0.1. Our results,shown in Table 2, indicate that there is indeed a relationship betweenthe starting m.o.i. and the final viral production. A starting m.o.i. ofless than 1 is optimal for the large scale manufacture of reovirus.

TABLE 2 Effect of M.O.I. on Viral Production (expressed as titer ± 95%CI in Log₁₀TCID₅₀/ml) TIME 1.0 MOI 0.5 MOI 0.1 MOI 24 HOURS 9.18 ± 0.368.55 ± 0.43 7.68 ± 0.24 36 HOURS 8.92 ± 0.52 9.30 ± 0.43 9.37 ± 0.48 48HOURS 9.55 ± 0.43 10.30 ± 0.37  9.68 ± 0.50 72 HOURS 9.55 ± 0.32 9.93 ±0.40 9.18 ± 0.40 96 HOURS 9.80 ± 0.00 10.30 ± 0.43  10.18 ± 0.36 

Furthermore, our results demonstrate that there is also an optimal timeat which to harvest the cells, which will be important. We found thatviral production is greatest after 24 hours. This is not surprising asprior to 24 hours, there would be insufficient time for adequate viralprotein synthesis and the assembly of the mature virion. More surprisingis the observation of a decrease in virus quantity at the 72 hour pointfollowed by a marked increase in the number of infectious particles atthe 96 hour time point. It is presumed that this slight decrease at 72hours is likely due to proteolytic degradation of the virus followed bya second round of virus replication at the 96 hour point.

We claim:
 1. A method of producing mammalian reovirus, comprising thesteps of: (a) contacting human embryo kidney 293 (HEK 293) cells with amammalian reovirus under conditions which result in reoviral infectionof said HEK 293 cells; (b) incubating the culture of said infected cellsfor a period of time sufficient to allow for viral replication; and (c)harvesting the virus produced.
 2. The method of claim 1 wherein themammalian reovirus is a human reovirus.
 3. The method of claim 2 whereinthe human reovirus is a serotype 3 reovirus.
 4. The method of claim 3wherein the serotype 3 reovirus is the Dearing strain.
 5. The method ofclaim 1 wherein the multiplicity of infection in step (a) is 10 or less.6. The method of claim 5 wherein the multiplicity of infection is 5 orless.
 7. The method of claim 6 wherein the multiplicity of infection is1 or less.
 8. The method of claim 7 wherein the multiplicity ofinfection is 0.5.
 9. The method of claim 8 wherein the multiplicity ofinfection is 0.1.
 10. The method of claim 1 wherein the virus isharvested when at least 5% of the cells in the culture remain viable.11. The method of claim 1 wherein the virus is harvested when 20-95% ofthe cells in the culture remain viable.
 12. The method of claim 11wherein the virus is harvested when 35-90% of the cells in the cultureremain viable.
 13. The method of claim 12 wherein the virus is harvestedwhen 50-80% of the cells in the culture remain viable.
 14. The method ofclaim 1 wherein the HEK 293 cells are cultured as adherent cells. 15.The method of claim 1 wherein the HEK 293 cells are cultured as asuspension.
 16. The method of claim 1 wherein the virus is harvested byseparating the cells from the culture media, disrupting the cells torelease the virus from the cells, and purifying the virus.
 17. Themethod of claim 16 wherein the cells are separated from the culturemedia by centrifugation and disrupted by freeze-thawing, and the virusis purified by a CsCl gradient.
 18. The method of claim 1, furthercomprising the step of freezing the harvested virus for storage.
 19. Themethod of claim 18 wherein the harvested virus is stored at −60° C. orbelow.
 20. The method of claim 1 further comprising the step oflyophilizing the harvested virus for storage.
 21. A method of producinginfectious reovirus comprising the steps of: (a) culturing human embryokidney 293 (HEK 293) cells in a culture medium containing 293 Serum FreeMedium supplemented with 4 mM L-glutamine at 36° C.±2° C., 6%±2% CO₂ and80%±5% relative humidity in spinner flasks at an impeller speed of 35-40rpm until the cells reach a cell density of about 10⁶ cells/ml; (b)infecting the cells with the Dearing strain reovirus at a multiplicityof infection of 0.5; (c) incubating the culture of said infected cellsin the same conditions as in step (a) until the percentage of viablecells drops to 50-80%; (d) harvesting the virus produced bycentrifugation of the culture, freezing-thawing to release the virusfrom the cells, and purifying the virus by a CsCl gradient; and (e)storing the virus at −60° C. or below.